1. Free radicals and antioxidants
What is „free radical“?
Reactive oxygen and nitrogen species (RONS)
Are the RONS always dangerous?
Well known term „oxidative stress“ - what is it?
Antioxidants - types and appearance
Markers of oxidative stress
Disorders Associated with Oxidative stress

2. Free radical - what is it?
Atom: proton, neutron, electronic shell (orbital)
Free radical
particles with an unpaired electron spinning around the nucleus. (can be atom, ions, molecule).
tend to reach equilibrium, plucks an electron from the nearest intact molecule.
most of biomoleculs are not radicals

3. Free radical and „science“
chemist during the thirties - there is superoxide
biochemist during the sixties - make a discovery of superoxid dismutase (SOD)
doctor free radicals are associated with many disorders

4. Mechanism of radical reactions
Radicals are highly reactive species
Three distinc steps
initiation (homolytic covalent bonds cleavage)
propagation (chain propagation)
termination

5. ROS (reactive oxygen species)
Free radicals
superoxide, O2 · -
hydroxyl radical, OH ·
peroxyl, ROO ·
alkoxyl, RO ·
hydroperoxyl, HO2 ·
Particals, which are not free radicals
hydrogen peroxide, H2O2 (Fenton´s reaction)
hypochlorous acid, HClO
ozone, O3
singlet oxygen, 1O2

7. RNS (reactive nitrogen species)
Particals, which are not free radicals
nitrosyl, NO+
nitrous acid, HONO
nitogen(III) oxide, N2O3
peroxynitrite, ONOO -
alkylperoxinitrite, ROONO
Free radicals
nitrogen(II) oxide, NO .
nitrogen(IV) oxide, NO2 .

8. The main sources of free radicals
membranes enzymes and/or coenzymes with flavine structures, hem coenzymes, enzymes containing Cu atom in an active site
1. respiratory chain mitochondria : mainly superoxide and then H2O2
approx 1- 4% O2 entres into resp. chain (mainly complexes I a III)

10. The main sources of free radicals II
2. Endoplasmic reticulum
superoxide creation (by cytochrome P- 450)
3. special cells (leukocytes)
superoxide creation by NADP-oxidas
4. hemoglobin to methemoglobin oxidation
(erytrocyte is „full“ of antioxidants)

11. Free radicals physiological function
Used by oxides a oxygenes
cytochromoxidase (toxic intermediates, H2O2 and superoxide, are bound to an enzymu)
monoxygenases - activate O2 in liver ER or in adrenal gland mitochondria ; hydroxylation

12. Free radicals physiological function II
ROS a RNS against bacteria
enzyme complex NADPH-oxidase of leukocytes
myeloperoxidase - catalysis of the following reaction
H2O2 + Cl- + H+ = HClO + H2O

13. Free radicals physiological function III
Signal molecules first messenger  second messenger  information net
This info net function is affected by the redox state of cells
redox state : antioxidant capacity, reduction equivalent availlability, RONS rate
 ROS: second messenger

14. Immunity vs. regulation
a massive production of ROS as immunity instrument x
an induction of the changes low concentration ROS, which are probably regulation mechanism

15. Antioxidant defence system
3 levels
inhibition of production the abundance of RONS
capture of radicals (scavengers, trappers, quenchers)
correction mechanism of destroyed biomoleculs

16. Antioxidants and scavengers review
1. Endogennous antioxidants
enzymes (cytochrome c,SOD, GSHPx, catalase)
nonenzymatic - fixed in membranes ( -tocopherol, -caroten, coenzym Q 10) - out of membranes (ascorbate, transferrin, bilirubin)

17. Antioxidants and scavengers review II
2. Exogennous antioxidants
FR scavengers
trace elements
drugs and compounds influence to FR metabolism

18. Enzymes defence mechanism

20. Superoxid dismutase (EC 1.15.1.1, SOD) 2O2. - + 2H+  H2O2 + O2
SOD - is present in all oxygen-metabolizing cells, different cofactors (metals)
an inducible in case of superoxide overproduction

21. Superoxid dismutase Mn 2+ SOD (SOD1) tetramer matrix mitochondria
lower stability then Cu, Zn - SOD

22. Superoxid dismutase Cu 2+/Zn 2+ SOD (SOD 2) dimer, Cu = redox centr
cytosol, intermitochondrial space
hepatocyt, brain, erytrocyte
high stability, catalysation at pH 4,5-9,5

23. Glutathion peroxidases
elimination of intracellular hydroperoxides and H2O2
2 GSH + ROOH  GSSH + H2O + ROH
cytosolic GSH - glutathionperoxidasa (EC , cGPx)
extracelullar GSH - glutathionperoxidasa (eGSHPx)
phospholipidhydroperoxide GSH - peroxidase (EC , PHGPx)

24. Catalasa (EC 1.11.1.6, KAT) 2 H2O2  2 H2O + O2
high affinity to H2O2 : peroxisomes hepatocytes mitochondria, cytoplasm of erytrocytes
tetramer with Fe, needs NADPH

25. High-molecula endogennous antioxidants
transferrin
ferritin
haptoglobin
hemopexin
albumin

26. Low-molecule endogennous antioxidats I
Ascorbate (vitamin C)
collagen synthesis
dopamine to epinephrine conversion
reduction agent
Fe absorption
antioxidant = reduction O2 · - OH ·, ROO·, HO2 ·
tocopheryl radical regeneration
prooxidant
Alfa-tocopherol a vitamin E
localise in membranes
produces hydroperoxides, which are changes by GSHPx

27. Ascorbic acid and its metabolites

28. Low-molecule endogennous antioxidats II
ubiquinone (coenzyme Q)
electron carrier in respisratory chain
co-operates with tocopheryl
carotenoides, -caroten, vitamin A
removing the radicals from lipids

29. Low-molecule endogennous antioxidats III
glutathione (GSH, GSSG)
in all mammalian cells (1-10 mmol/l)
important redox buffer
2 GSH  GSSG + 2e- + 2H+
ROS elimination, stabilisation in reduction form ( SH- groups, tocopheryl and ascorbate regeneration)
substrate of glutathione peroxidases

30. Low-molecule endogennous antioxidats IV
Lipoic acid (lipoate)
PDH cofactor tocopheryl and ascorbate regeneration
melatonin
lipophilic ; hydroxyl radicals scavenger

31. Low-molecule endogennous antioxidats V
uric acid (urates)
bilirubin
flavonoids

32. Trace elements influence to FR metabolism
Selenium
influence to vitamin E resorption, part of selenoproteins  of Se = insufficient immun. respons, erytrocytes hemolysis, methemoglobin synthesis
Zinc
cell membrane stabilisation Fe antagonist

33. Be carefull - this equilibrium can be disbalance in both sides
Oxidative stress
Equilibrium failure between creation and a elimination of RONS leads to
oxidative stress
Be carefull - this equilibrium can be disbalance in both sides

34. Oxidative damage to lipid
unsaturated bonds loss
arising of reactive metabolites (aldehydes)
Sequel
changes in fluidity and permeability of membranes
membranes integral enzymes are influenced

35. The peroxidation of linoleic acid

36. Oxidative damage to proteins
agregation, fragmentation and cleveage
reaction with hem iron ion
functional group modification
Sequel
changes in: enzymes activity, ions transport
proteolysis

37. Oxidative damage to DNA
saccharide ring cleveage
bases modification
chain breakeage
Sequal
mutation
translation mistakes
protoesynthesis inhibition

38. Oxidative stress markers
Free radicals detection
very difficult, because of chem-phys. properties
Oxidative stress products detection
more simple, a wide range of techniques

39. Oxidative stress markers II
Lipoperoxidation markers:
malondialdehyde (MDA), conjugated diens, isoprostanes
Oxidative damage to protein markers :
protein hydroperoxides
Oxidative damage to DNA :
modified nucleosides

40. Antioxidants determination
ascorbate
tocopheryl
SOD
GSHPx
glutathion

41. Disorders Associated with Oxidative stress
Neurological
Alzheimers Disease
Parkinson‘s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis

42. Disorders Associated with Oxidative stress
Others conditions
Obesity
Air Pollution
Toxicity
Inflammation